Correlation of Visual System Biomarkers With Motor Deficits in Experimental Autoimmune Encephalomyelitis-Optic Neuritis

Abstract

Purpose: Experimental autoimmune encephalomyelitis (EAE) scoring, the most commonly used primary outcome metric for an in vivo model of multiple sclerosis (MS), is highly variable and subjective. Here we explored the use of visual biomarkers in EAE as more objective and clinically relevant primary outcomes.

Methods: Motor impairment in myelin oligodendrocyte glycoprotein-immunized C57BL/6J mice was quantified using a five-point EAE grading scale. Pattern electroretinography (pERG) and retinal ganglion cell/inner plexiform layer (RGC/IPL) complex thickness were measured 60 days after induction. Optic nerve histopathology was analyzed at endpoint.

Results: EAE mice displayed motor impairments ranging from mild to severe. Significant correlations were seen between pERG amplitude and last EAE score, mean EAE score, and cumulative EAE score. Optical coherence tomography (OCT) analysis demonstrated a significant correlation between thinning of the RGC/IPL complex and both EAE score and pERG amplitude. Optic nerve histopathology showed significant correlations between demyelination and cumulative EAE score, pERG amplitude, and RGC/IPL complex thickness, as well as between immune cell infiltration and cumulative EAE score, pERG amplitude, and RGC/IPL complex thickness in EAE mice.

Conclusions: Unlike EAE scoring, pERG and OCT show direct measurement of retinal structure and function. Therefore we conclude that visual outcomes are well suited as a direct assessment of optic nerve involvement in this EAE model of MS while also being indicative of motor impairment.

Translational Relevance: Standardizing directly translatable measurements as primary outcome parameters in the murine EAE model could lead to more rapid and relevant testing of new therapeutic approaches for mitigating MS.

Introduction

Multiple sclerosis (MS) is a multifaceted autoimmune disorder characterized by axonal damage, gliosis, and inflammatory demyelination within the central nervous system (CNS).¹ More than 2.5 million individuals are affected by MS worldwide making it a leading cause of atraumatic neurological disability in young adults.^2,3 Acute optic neuritis (ON), inflammation of the optic nerve, occurs in approximately 21% to 45% of newly diagnosed cases, where it typically presents with a range of visual disturbances, including blurred vision, reduced color perception, and retrobulbar pain.^4–7 

Declines in the visual system because of MS extend beyond clinical symptoms and include structural and functional alterations detectable via noninvasive methods including pattern electroretinography (pERG) recordings and optical coherence tomography (OCT) imaging. pERG records the electrical signal produced by the retina as a function of time in response to a stimuli of a contrast-reversing checkerboard pattern and is typically recorded noninvasively from the lower eyelid or cornea.^8,9 MS disease progression has been associated with reductions in pERG amplitude.^10,11 OCT has emerged as a valuable non-invasive tool for assessing retinal structures in individuals with MS.¹² More precisely, OCT enables high-resolution, cross-sectional imaging of the retina, allowing for precise measurements of the retinal nerve fiber layer (RNFL) and the inner plexiform layer (IPL), collectively representing retinal ganglion cell (RGC)/IPL complex thickness.^13,14 Therefore changes visualized in the RNFL are the result of retrograde degeneration from lesions in the optic nerve, chiasm, or visual tract.¹⁵ Thinning of the RNFL and RGC/IPL complex has been associated with MS, particularly when evaluated over time.^16–19 Thus, OCT provides an opportunity to detect subtle changes in retinal structure associated with MS,^13,17 making it an essential tool for studying the relationship between CNS involvement and peripheral manifestations such as visual deficits.²⁰ Petzold et al.²¹ conducted a meta-analysis of 5776 MS eyes and found that not only was peripapillary RNFL thickness reduced in both MS ON eyes and MS non-ON eyes relative to healthy control eyes, but there was also a pronounced thinning of the ganglion cell layer and IPL in both MS ON eyes and MS non-ON eyes when compared to control eyes, thereby demonstrating that neuroaxonal injury is measurable by OCT. These findings demonstrate the value of OCT as a robust and reproducible tool for monitoring MS progression, and that the visual system is a potential surrogate marker for disease progression in MS. 

Experimental autoimmune encephalomyelitis (EAE) is a well-established animal model of MS.^22,23 EAE mimics relevant pathological features of MS, including immune-mediated demyelination, axonal injury, and inflammatory responses within the CNS, as well as ON.^24,25 Thus EAE is a valuable tool for exploring connections between CNS dysfunction and sensory manifestations such as visual impairments. Current primary outcomes largely depend on EAE scoring, an assessment of impaired motor function using a five-point grading scale,^25–28 and are ultimately related to inflammation of the spinal cord. With regard to the visual phenotype of MS/EAE, functional and structural outcomes would be less subjective and more directly confirmatory of optic nerve inflammation, demyelination, and axonal damage. 

Optic nerve histopathology offers an excellent method for discovering structural alterations in the visual system of the EAE model. Specifically, the ability to detect and quantify demyelination of the axon and immune cell infiltration using readily available lab techniques, such as Luxol fast blue (LFB) and hematoxylin and eosin (H&E) staining.²⁹ The optic nerve, as a key component of the marvelously complex afferent visual system, is unique in that it is directly and only related to visual structure and function, whereas other CNS tissues such as the spinal cord are affected by inputs from multiple body systems.^30–32 

This study aims to bridge the gap in our understanding of the relationships between structural/functional changes in the visual system and the wider spectrum of motor deficits observed in EAE. By inducing EAE in a murine model and closely monitoring clinical progression, we intend to determine whether alterations in the visual system, including pERG amplitude, and RGC/IPL complex thinning as measured by OCT, correlate with motor impairments and structural alterations of the optic nerve. These investigations not only hold the potential to enhance our understanding of the interrelation between structural, functional, and imaging parameters of the retina in the context of murine EAE and MS but also offer insights into the feasibility of using noninvasive visual assessments, particularly OCT, as potential indicators of CNS involvement. OCT is suitable for evaluating the potential treatment effects of new drugs in preclinical settings because it has been previously used as a primary and secondary clinical outcome.^33–36 

Here we demonstrate significant correlations between overall motor impairment, morphological alterations of the optic nerve, visual function, and structural changes in the retina of EAE mice. These correlations underscore the promises of pERG and OCT as critical tools for understanding the relationship between the visual system and motor deficits in the context of murine EAE and its potential application in MS research and clinical practice. 

Methods

Experimental Design and EAE Model

All animal experiments were approved by the local IACUC and conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Eight- to 12-week-old female C57BL/6J mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and housed in 12-hour light-dark cycle (735 lux) with food and water as desired. After a suitable period of adaptation to the facility, EAE-ON was induced as previously described.^28,29,37,38 Briefly, 15 mice were injected subcutaneously with 200 µg of MOG_35–55 (Sigma Aldrich, St Louis, MO, USA) emulsified with complete Freund's adjuvant (Sigma Aldrich) containing 2 mg/mL of Mycobacterium tuberculosis H37Ra (BD Difco, Franklin Lakes, NJ, USA) at each of two locations proximal to the shoulder blades. The animals were then injected intraperitoneally with 400 ng of pertussis toxin (Sigma Aldrich), and then again with the same amount two days later. Because no obvious ophthalmic differences between sham-immunized and naïve mice have been reported,^39,40 a cohort of age-matched, nonimmunized mice (n = 15) was implemented as controls (CTRL). As previously described,^25–28 all mice were scored daily postinduction for motor impairment. Briefly, clinical progression was assessed using a five-point EAE grading scale: 0 = no symptoms, 0.5 = partial tail paralysis, 1 = tail paralysis, 1.5 = partial tail paralysis and waddling gait, 2 = tail paralysis and waddling gait, 2.5 = partial paralysis of one or both hind limbs, 3 = paralysis of one limb, 3.5 = paralysis of one limb and partial paralysis of another, 4 = paralysis of two limbs, 4.5 = moribund state, and 5 = death. OCT and pERG were recorded at day 60 postinduction. At the end of the study, all animals were euthanized by CO₂ inhalation followed by cervical dislocation and optic nerve tissue harvest. Cumulative EAE was calculated as the sum of all EAE scores for a given animal. 

Pattern Electroretinography

PERG was completed on day 60 postinduction using a JORVEC system (Intelligent Hearing Systems, Miami, FL, USA), as previously described.^41,42 After overnight dark adaptation, mice were anesthetized via intraperitoneal injection of ketamine (33 mg/kg; Mylan, Canonsburg, PA, USA), xylazine (2.5 mg/kg; Akorn Inc., Lake Forest, IL, USA) and acepromazine (0.75 mg/kg; Rattlesnake Drugs, Scottsdale, AZ, USA), and pupils were dilated with 1% tropicamide (Akorn Inc.). Genteal gel (Alcon Laboratories, Geneva, Switzerland) was applied to the corneal surface to maintain corneal integrity. Constant body temperature was maintained between 37° C and 38° C throughout the procedure using the system's integral heat pad. Both eyes were simultaneously assessed, and 372 pERG traces were recorded under mesopic conditions between 1.7 lux (no pERG running) and 100–118 lux while monitors displayed pERG stimuli. The pERG stimuli contained six bars (three white and three dark, 0.05 cycles/deg) alternating black and white horizontal patterns presented on two 15 × 15 cm tablets at 0.992 Hz for the right and 0.984 Hz for the left eyes at maximum contrast (98%). Cutoff filter frequencies were set to 1 to 300 Hz. The averaged amplitudes were measured in microvolts (µV) from the zero line to the N1 trough (N1 amplitude), from the N1 trough to the P2 peak (N1 to P2 amplitude), and from the P2 peak to the N2 trough. Data displaying low or atypical pERG waveforms or nonunique peak identification were excluded from statistical analysis. 

Optical Coherence Tomography

OCT imaging was performed as previously described.^28,29,38,43 Mice were anesthetized using 2% to 4% isoflurane (Baxter, Deerfield, IL, USA) supplied via a SomnoSuite system (Kent Scientific, Torrington, CT, USA). Their pupils were dilated using 1% tropicamide (Alcon Laboratories). Corneal integrity was preserved via the addition of Genteal gel (Alcon Laboratories) to the corneal surface. Retinal images were then obtained via a Bioptigen Envisu OCT device (Bioptigen, Morrisville, NC, USA). Volume scans (1.4 × 1.4 × 1.57 mm) centered at the optic nerve head were obtained. Measurements of the RNFL and the IPL were obtained as previously described.^28,43 Briefly, borders of the RNFL and the IPL were determined manually, and thickness between the borders of the RNFL from the inner limiting membrane to the outer borders of the IPL (referred to as the RGC/IPL complex) was measured 400 µm away from the center of the optic nerve head in the superior, inferior, nasal, and temporal quadrants. Data from eyes with cold cataract formation or indistinguishable retinal layer borders were excluded from statistical analysis. 

Optic Nerve Histopathology

Optic nerves were harvested at the end of the study from both control and EAE mice, fixed in 4% paraformaldehyde, and embedded in paraffin. Seven-micrometer-thick longitudinal sections were cut and stained with LFB or LFB and H&E. Sections were then graded for demyelination and for immune cell infiltration. Demyelination was graded using a three-point scale with the following criteria: 0 = normal optic nerve, fully myelinated; 1 = scattered foci of demyelination; 2 = prominent foci of demyelination; and 3 = large areas of demyelination.^28,29 Immune cell infiltration was graded on a four-point scale where 0 = no cellular infiltration, 1 = mild infiltration, 2 = moderate infiltration, 3 = severe infiltration, and 4 = profound cellular infiltration.^29,44,45 

Statistical Analysis

All statistical analyses were conducted using GraphPad Prism v9.4 (GraphPad Software, San Diego, CA, USA). P values < 0.05 were considered statistically significant. All numerical data such as pERG and OCT results passed testing for standard distribution using Shapiro-Wilks test and differences between EAE mice and controls were analyzed using a two tailed t-test. OCT and pERG values are expressed as mean ± standard error of the mean. Ordinal data for grading optic nerve demyelination and infiltration were analyzed via a two-tailed Mann-Whitney test. Intereye differences were assessed using Bland-Altman analysis and results are reported as percent differences from the average including the bias values. Correlation analysis of numeric data was conducted using Pearson r. Spearman r test was used for correlation analysis that compares ordinal to numerical values. Data in correlation graphs are expressed as individual data points per EAE animal/eye and the best-fit trendline is displayed in graphs. 

Results

Evaluation of Motor Impairment

Grading of motor impairment is currently the most common method for evaluating EAE progression. First signs of motor impairment were observed in five of 15 EAE mice at day 10 after EAE induction (Fig. 1). At day 15, all EAE-induced mice had positive EAE grading scores. EAE peak scores were noted between day 14 and day 23 at which 12 out of 15 EAE mice had a score ≥2. All of these EAE mice showed signs of recovery (lower scores) within 4.75 ± 3.5 days after their EAE peak. Moderate overall severity of clinical symptoms with a relatively high degree of variation was observed in EAE mice over time. EAE scores range from 0.5–4 with a mean EAE score of 1.8 ± 0.7, which corresponds to an average cumulative EAE score of 109 ± 43. Of note is the large heterogeneity in this EAE cohort, normally undesirable for a research study, but that is ideal for a correlation analysis. Individual EAE mice presented with a range of motor impairments ranging from mild to severe, as demonstrated by last EAE score, mean EAE score, and cumulative EAE score (Table). 

Table.

 

Individual EAE Mice Presented With a Range of Motor Impairments Ranging From Mild to Severe. EAE Scores Range from 0.5–4 With a Mean EAE Score of 1.8 ± 0.7, Which Corresponds to an Average Cumulative EAE Score of 109 ± 43. Mice Are Ordered by Increasing Severity for Ease of Reference

 

Figure 1.

 

EAE scoring confirms motor impairment in EAE mice. EAE was induced in 15 female C57BL/6J mice by immunization with MOG_33–55, complete Freund's adjuvant, and pertussis toxin. EAE mice were scored daily postinduction for motor sensory impairment using a five-point grading scale. Motor impairment was observed in EAE mice beginning around day 10 postinjection. EAE peak scores were noted between day 14 and day 23 at which 12 out of 15 EAE mice had a score ≥2. All of these EAE mice showed signs of recovery (lower scores) within 4.75 ± 3.5 days after their EAE peak. Moderate overall severity of clinical symptoms with a relatively high degree of variation was observed in EAE mice. Each datapoint represents the mean ± standard error of the mean for all 15 EAE animals.

Reduction of Retinal Function Measured by pERG

A decline in pERG amplitude is representative of RGC dysfunction. Thus we examined changes in pERG 60 days postinduction. PERG amplitude was measured from P1 peak to N2 trough (Fig. 2A). Traces of two eyes, one from an EAE animal with an atypical waveform including two P1 peaks at an amplitude of 8.7 µV and one from a control mouse with an unusually high amplitude of 36 µV, were excluded from subsequent analysis. Fourteen eye pairs for each group were used to determine intereye differences. Results show good overall consistency in magnitudes of OS/OD amplitudes (Fig. 2B) but a high intraindividual variability of up to 48% from the average and a bias of 1.9 in naive controls and 50% with a bias of 1.5 in the EAE group respectively. Because there were no statistical differences in pERG data between OS and OD eyes (P = 0.83), we subsequently included values from animals where we had measurements from only one eye for the comparison of EAE versus controls. pERG data from EAE mice (n = 15) demonstrated a significantly decreased amplitude relative to healthy controls (n = 15; Fig. 2C; EAE mean 16.2 ± 0.90 µV vs. CTRL mean 23.7 ± 0.75 µV; P < 0.0001). Correlation analysis of pERG amplitudes between OS and OD eyes using data from both groups revealed a robust and highly significant relation between eye pairs, as well as a distinct separation between the EAE group from naïve controls (Fig. 2D; r = 0.64; P = 0.0003). Furthermore, a significant negative correlation was observed in EAE mice between P1 to N2 amplitudes and last EAE score, mean EAE score, and cumulative EAE score (Figs. 2E, 2F). 

Figure 2.

 

PERG demonstrates reduction of retinal function in EAE mice. (A) On day 60 postinduction, pERG amplitude (A) was measured from P1 peak to N2 trough. (B) Analysis of intereye relation of EAE mice (n = 15; gray dots) and 15 age-matched healthy control mice (CTRL; gray rectangles) showed notable intereye variability but no significant differences between OS versus OD group averages. (C) EAE mice demonstrated a significantly decreased averaged amplitude obtained from both eyes when compared to averaged pERG amplitudes from controls. Data are displayed as mean ± standard error of the mean. (D) Regression analysis revealed a strong within-mouse, intereye correlations for both the EAE group and the naïve control cohort. (E) Significant correlations were observed in EAE mice between P1 to N2 amplitude and last EAE score), (F) mean EAE score, and (G) cumulative EAE score. Each datapoint represents the average pERG amplitude values of both eyes of each EAE mouse.

Notable Thinning of Retinal Layers Detected by OCT

Thinning of the RGC/IPL complex, as measured by OCT, is indicative of loss of retinal structure and is associated with declining retinal function. The RGC/IPL complex was measured from the top of the RNFL to the bottom of the IPL (Fig. 3A). OCT images could not be obtained from one EAE animal (both eyes) because of cold cataract formation. Data was also excluded from one eye each of another two EAE mice because of insufficient retinal layer segmentation. We determined there were no intereye differences with only marginal variability of <6% from the group average with a bias of 0.03 in the control cohort and a bias of 0.3 with only 12 eye pairs in the EAE group (Fig. 3B). Similar to the pERG analysis, we included the average RGC/IPL complex thickness of all 15 controls and used the average of 12 EAE eye pairs, substituted with the single eye RGC/IPL complex thickness measurements of another two EAE mice for the group comparison. EAE mice demonstrated a significant thinning of the RGC/IPL complex when compared to healthy controls (Fig. 3C; EAE mean 58.79 ± 1.09 µm vs. CTRL mean 66.27 ± 0.88 µm; P < 0.0001). We also found a significant correlation between OS and OD eye RGC/IPL complex thickness in both EAE mice and naïve controls (r = 0.92; P < 0.0001, Fig. 3D). In EAE mice, significant negative correlations were observed between RGC/IPL complex thickness and last EAE score, mean EAE score, and cumulative EAE score (Figs. 3E–G), whereas a significant positive correlation was seen between RGC/IPL complex thickness and pERG P1 to N2 amplitude (Fig. 3H; r = 0.5; P = 0.0072). 

Figure 3.

 

OCT indicates reduction of retinal structure in EAE mice. On day 60 postinduction, retinal images were obtained from anesthetized mice via OCT. (A) The boundaries of the RGC/IPL complex, shown in yellow, were measured from the top of the retinal nerve fiber layer to the bottom of the IPL. Extremes of retinal thickness are demonstrated for ease of visualization. (B) Analysis of intereye analysis demonstrated a low RGC/IPL complex thickness variability between OS and OD eyes in individual CTRL and EAE mice. (C) OCT analysis revealed significant thinning of the RGC/IPL complex in EAE mice when compared to CTRLs. (D) Despite significant RGC/IPL thinning in EAE mice, a strong within-mouse, intereye correlation was observed for both the EAE and naïve control cohort. Furthermore, significant correlations were observed (E) between RGC/IPL thickness and last EAE score, (F) mean EAE score, and (G) cumulative EAE score. Analysis of functional and structural relationships revealed a significant correlation between the decline in the pERG amplitude and thinning of the RGC/IPL complex in EAE mice. In graphs (B–G), each datapoint represents an average of the RGC/IPL complex thickness for both eyes, whereas each datapoint in (H) represents the RGC/IPL complex thickness and P1 to N2 amplitude of an individual eye.

Severe Disruption of Optic Nerve Structure

Optic nerve histopathology provides the ability to quantify optic nerve structure, which can be reasonably extrapolated to the status of optic nerve function. Thirteen optic nerve pairs plus 2 single optic nerves from controls and 12 pairs plus 3 single optic nerves from EAE mice were used for histopathology. Sections were scored for demyelination and infiltration (Fig. 4A). In accordance with our pERG and OCT results, no intereye differences were observed in the EAE group with respect to demyelination (OS vs. OD, P = 0.63) or immune cell infiltration (OS vs. OD, P = 0.43). Similarly, no deviations in the grade of demyelination (OS vs. OD, P = 0.9) or cell infiltration (OS vs. OD, P = 0.57) was noted in the control cohort, and we further determined significant intereye correlations for both histopathologic parameters in both groups (demyelination r = 0.77, P < 0.0001 and cell infiltration r = 0.89, P < 0.0001). As expected, optic nerves obtained from EAE mice showed a significant increase in both demyelination (Fig. 4B; EAE median 2 vs. CTRL median 0.5; P < 0.0001) and cell infiltration (Fig. 4C; EAE median: 2.5 vs. CTRL median 0; P < 0.0001) relative to healthy controls. For optic nerves from EAE mice, significant correlations were observed between demyelination grade and cumulative EAE score (Fig. 4D; r = 0.64; P = 0.009), pERG P1 to N2 amplitude (Fig. 4E; r = −0.55; P = 0.0036), and RGC/IPL complex thickness (Fig. 4F; r = −0.60; P = 0.0018). Furthermore, in this same group, significant correlations were observed between immune cell infiltration grade and cumulative EAE score (Fig. 4G; r = 0.80; P = 0.0003), pERG P1 to N2 amplitude (Fig. 4H; r = −0.49; P = 0.011), and RGC/IPL complex thickness (Fig. 4I; r = −0.72; P < 0.0001). 

Figure 4.

 

Optic nerve histopathology indicates structural degradation of optic nerve in EAE mice. (A) Optic nerve sections from both CTRL and EAE mice were stained with LFB or LFB and H&E. As expected, optic nerves obtained from EAE mice showed a significant increase in both (B) demyelination and (C) immune cell infiltration relative to healthy controls. Also, significant correlations were observed between demyelination and (D) cumulative EAE score, (E) pERG P1 to N2 amplitude, and (F) RGC/IPL complex thickness. Significant correlations were also observed between immune cell infiltration and (G) cumulative EAE score, (H) pERG P1 to N2 amplitude, and (I) RGC/IPL complex thickness. In (B), (C), (D), and (G), each datapoint represents an average for both eyes, whereas each datapoint in (E), (F), (H), and (I) represents an individual eye.

Discussion

The significant correlations observed via histopathology are particularly interesting as they allow an indirect comparison between the various in vivo measurements. It is noteworthy that the same correlations were observed using both histopathological grading scales (infiltration and demyelination) when compared to all in vivo outcomes: last EAE score, mean EAE score, cumulative EAE score, pERG amplitude, and RGC/IPL thickness as measured by OCT. 

EAE scoring is directly indicative of motor impairment and, thus far, is the most common in vivo outcome parameter. The onset of motor impairment at day 10 and its peak at around day 20 are typical of our own prior observations and those of others.^25,28,46,47 What was more unusual was the wide range of cumulative EAE scores observed. Previously described induction regimes^28,29,46 generally display more uniform levels of motor impairment, which is preferable for experimental purposes. However, the higher range of EAE scores observed in this study was ideal for correlation analysis of the relationships between disease progression and visual system parameters. Other groups have reported considerable heterogeneity in the EAE phenotype with some observing unilateral optic neuritis or bilateral involvement at different magnitude.^45,48,49 Although this differs from our laboratory's typical observations, it serves to further highlight the need for more objective disease markers. A considerable disadvantage of EAE scoring is its reliance on a subjective judgement of the animals’ condition. This opens the possibility that the observer's skill level can potentially influence the outcome of the data, especially during the assessment of potentially beneficial effects of new drug candidates versus a placebo EAE group. Thus including additional less subjective and highly translatable outcome measurements would not only serve as confirmation of EAE scores but also would significantly increase the scientific rigor of EAE studies. 

PERG amplitude, as a direct measure of the electrical output of RGC, represents a minimally invasive test of visual system function.⁹ Furthermore, it has the advantage of producing objective data not immediately dependent on the interpretation of the operator.⁵⁰ Although not directly indicative of motor impairment, it was noteworthy that the decline in pERG amplitude in our EAE animals showed highly significant correlation to motor impairment, as well as to optic nerve histopathology and RGC/IPL complex thinning. Thus this method is not only more directly relevant for evaluation of ON-induced RGC degeneration, but it can also provide an excellent correlation to the overall state of disease progression in EAE animals. The advantage of using pERG is that it is directly clinically applicable because it is used to measure the status of patients’ ON function and might be used as a prognostic marker for progression. In recurrent MS-ON, pERG amplitude is significantly reduced compared to control eyes, as seen in our EAE model.⁵¹ Disadvantages of this method include the need to account for factors such as variations in eye size or electrode position, extended preparation time, and the potential that the location of the electrodes at the cornea could cause irritation.^52,53 All pERG data presented in this study were collected as end timepoint measurements. It is probable that pERG changes are detectable before structural deficits become evident; however, ketamine/xylazine anesthesia is associated with increased mortality in weakened mice.^54,55 Although not reported in the literature, researchers in the EAE field are aware of this high ketamine/xylazine anesthesia-related mortality, which makes it difficult to obtain longitudinal pERG data at earlier timepoints without losing the EAE animal, and thus the opportunity for subsequent end-point measurements. Although we cannot currently answer the question of whether pERG changes precede RGC/IPL thinning, it would be an interesting question for subsequent studies. 

Measurement of RGC/IPL complex thickness via OCT allows a direct, objective, and noninvasive measure of the physical structure of the retina, which can be extrapolated to visual system function. Thinning of the RGC/IPL complex is known to correspond to deterioration of vision, as well as disease progression in MS and associated diseases.^35,56,57 In this study, we observed notable thinning of the RGC/IPL complex in EAE mice when compared to naïve control mice. This thinning correlated strongly with motor deficits and optic nerve demyelination and cell infiltration, demonstrating that OCT measurements provide an excellent objective indicator of the visual phenotype and progression in the EAE model on the structural level. Our observations are complementary to the work of Cruz-Herranz et al.²⁴ and Mey et al.,⁵⁸ which not only demonstrated a loss of RGC and retinal thinning in MOG-induced EAE mice compared to sham-immunized controls or naïve mice, respectively, but also a significant correlation of RGC loss and decrease of inner retinal thickness. Additionally, both studies showed that retinal thinning in EAE mice was associated with EAE severity.^24,58 A particular strength of this minimally invasive analysis of the retinal structure is that it is directly translatable to the clinic, because OCT has become the most widely used ophthalmic imaging method for human patients in the developed world.⁵⁹ A recent study by Rosenkranz et al.⁶⁰ used OCT to assess retinal thickness in primary progressive MS patients and found that OCT (as a component of advanced vision testing) is a promising prognostic marker. Furthermore, OCT is a routine procedure within most human clinics and follows established guidelines and protocols for MS ON, which allows for consistent and objective measurements. The lack of guidelines for OCT measurements in rodents, particularly EAE ON, is a challenge that has not yet been adequately addressed. Future efforts should be made to determine the precise methodology that best reflects clinical practice and to standardize this methodology across the ON research field, as has previously been done by Hecker et al. for optomotor response, another clinically relevant technique.⁴⁴ 

In this study, we have demonstrated a significant correlation between visual system structure and function and overall motor impairment in EAE animals. Thus it seems appropriate to use visual outcomes, particularly OCT and pERG, as direct measurements of ON-related structural decline. Furthermore, the widespread use of these directly translatable measurements as primary outcome parameters, particularly in conjunction with continued efforts to align OCT protocols used in EAE studies with those being used for MS patients, will aid in the immediate relevance of future research. The use of OCT for measuring disease progression over time in both clinical settings and EAE is highly relevant for drug testing. However, future EAE-ON studies are needed to determine whether the results of our correlation analysis are consistent in EAE-ON animals that have received various forms of treatment. Ultimately, this may lead to a more accurate assessment of EAE progression by removing the subjectivity of EAE grading between investigators. It may also lead to a more accurate understanding of the effectiveness of treatments being investigated. Optomotor response and OCT provide sufficient data to analyze visual function in EAE, particularly if paired with pERG or visual evoked potentials and thus are more objective and better translatable to the clinic than EAE scoring. Because EAE score is not directly applicable to the clinic, motor sensory impairment should be considered secondarily to the visual pathway in assessing clinical relevance. 

Acknowledgments

Supported by Merit Grant 1I01RX002978-01 and Center Grant 1I50RX003002-01 from the United States (U.S.) Department of Veterans Affairs Rehabilitation Research and Development Service. 

Disclosure: B.W. Elwood, None; C.R. Godwin, None; J.J. Anders, None; R.H. Kardon, None; O.W. Gramlich, None